There are >200 million malaria cases and ˜1 million deaths per year caused by Plasmodium falciparum (Pf) (World Health Organization. Global Malaria Programme. World Malaria Report 2010: World Health Organization, 2010; Murray C J, et al. Global malaria mortality between 1980 and 2010: a systematic analysis. 2012 Lancet 379: 413-431). Recently, progress has been made controlling malaria due to investment of billions of dollars in the use of bednets, insecticides, and drugs. However, as highlighted in a recent editorial (Editorial: Malaria 2010: More Ambition and Accountability Please 2010 Lancet 375:1407), a commercially available malaria vaccine is still badly needed. To prevent infection, disease, and transmission an ideal single stage vaccine should target the pre-erythrocytic (sporozoite and liver) stages (Plowe C. V. et al. The potential role of vaccines in the elimination of falciparum malaria and the eventual eradication of malaria 2009 J. Infect. Dis. 200:1646-1649; Alonso P. L. et al. A research agenda for malaria eradication: vaccines. 2011 PLoS Med 8: e1000398). Such a vaccine would have hμge public- and private-sector markets. In public-sector markets it would be used in infants, young children, and adolescent females (preventing malaria during pregnancy) and for entire populations for geographically focused malaria elimination campaigns (Id.). Individuals from non-malarious countries who spend time in areas with malaria (travelers, military, government officials, students, business people, etc.) and middle and upper class residents of countries with malaria comprise the private-sector market.
Data indicating a highly effective vaccine might be possible came from trials in which volunteers immunized by the bites of mosquitoes infected with radiation-attenuated Pf sporozoites had high-level (>90%), sustained (≧10 months) protection against experimental challenge (Hoffman, S. L., et al. 2002 J. Inf. Dis. 185:1155-64).
It has been shown that a vaccine incorporating live attenuated Pf sporozoites can be manufactured (SANARIA™ PfSPZ Vaccine). This process has recently been described (Hoffman S. L. et al. Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria. 2010 Hum. Vac. 6:97-106. DOI: 10396 [pii].). The ability of PfSPZ Vaccine to induce antigen-specific immune responses in humans was also demonstrated (Epstein, J. E., et al. Live Attenuated Malaria Vaccine Designed to Protect Through Hepatic CD8+ T Cell Immunity 2011 Science 334 (6055):475-480).
It is thought that an attenuated PfSPZ vaccine delivered by a parenteral non-intravenous route and capable of demonstrating a protective efficacy comparable to that achieved with PfSPZ administered IV would require large numbers of sporozoites and a multi-dose regimen. In a mouse model, present data suggests that approximately 7 times as many Plasmodium yoelii (Py) sporozoites administered intradermal (ID) or subcutaneous (SC) are required compared to IV administration in order to achieve >80% protection in mice (Epstein, et al., Id.). A more promising approach for the development of a highly effective parenteral non-IV vaccine would likely include the use of an adjuvant.
The adjuvant: A glycolipid adjuvant that stimulates natural killer T-cells (NKT) was identified in mice. (Gonzalez-Aseguinolaza G, et al. Natural killer T cell ligand α-galactosylceramide enhances protective immunity induced by malaria vaccines 2002 J. Exp. Med. 195: 617-624; U.S. Pat. No. 7,534,434). Using a single IV-administered dose of radiation attenuated P. yoelii sporozoites (suboptimal for protection) it was demonstrated in the Gonzalez-Aseguiniola paper that distal intraperitoneal (IP) administration of α-galactosylceramide (α-GalCer), a ligand for natural killer T (NKT) cells, could induce a higher degree of protection (>90%), than IV administration of irradiated P. yoelii sporozoites alone, which conferred only 20% protection.
Natural Killer T (NKT) cells are a subset of T cells that co-express receptors of T cell and NK cell lineages and recognize their cognate antigen presented by the MHC-like CD1d on antigen presenting cells (APCs). The major subset of NKT cells are distinguished by their restricted expression of an invariant TCR (invTCR) and are termed iNKT cells. The increased potency of IV administered sporozoites and distally administered IP adjuvant described in Gonzalez-Aseguinolaza et al. correlated with enhanced IFN-gamma secretion by CD8+ T cells and was dependent on iNKT cells and CD1d (Id.). This first-identified iNKT TCR ligand, α-GalCer, extracted from the Agelas mauritianus sea sponge, was discovered while screening for compounds with anti-tumor activity. It has a high affinity for CD1d, is a potent activator of iNKT cells in both mouse and human, and has been used extensively to study the function of iNKT cells (Brossay L, et al. CD1d-Mediated Recognition of an A-Galactosylceramide by Natural Killer T Cells is Highly Conserved through Mammalian Evolution 1998 J. Exp. Med. 188:1521-1528; Kobayashi E, et al. KRN7000, A Novel Immunomodulator, and its Antitumor Activities 1995 Oncol. Res. 7: 529-534; Kawano T, et al., CD1d-restricted and TCR-mediated activation of vα14 NKT cells by glycosylceramides 1997 Science 278: 1626-1629. In vivo administration of α-GalCer in mice results in a cascade of events beginning with signaling through the invTCR by APCs expressing CD1d. Macrophages, dendritic cells, B cells, Kupffer cells in the liver, and hepatocytes all have constitutive expression of CD1d (Mandal M, et al., Tissue distribution, regulation and intracellular localization of murine CD1 molecules 1998 Mol. Immunol. 35: 525-536; Brossay L., et al., Mouse CD1 is mainly expressed on hemopoietic-derived cells 1997 J. Immunol 159: 1216-1224; Roark, J. H., et al., A. CD1.1 expression by mouse antigen-presenting cells and marginal zone B cells 1998 J. Immunol. 160: 3121-3127).
Stimulated iNKT cells rapidly secrete pre-stored cytokines (unlike traditional T cells) that reciprocally activate APCs (Tomura M., et al., A novel function of Vα14+CD4+NKT cells: stimulation of IL-12 production by antigen presenting cells in the innate immune system 1999 J. Immunol. 163: 93-101; Fujii, S., et al., Activation of natural killer T cells by α-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein 2003 J. Exp. Med. 198: 267-279) enhancing their ability to prime CD4+ and CD8+ T cells (Fujii, S., et al., The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation 2004 J. Exp. Med. 199: 1607-1618; Hermans, I. F., et al., NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells 2003 J. Immunol. 171: 5140-5147) to generate a powerful cell-mediated immune response. In the paper of Gonzales-Aseguinolaza et al., supra, the legend of FIG. 2A states that a group of BALB/c mice was immunized subcutaneously with irradiated sporozoites [P. yoelii] together with or without administration of α-GalCer by the same route, and when splenic lymphocytes were isolated and the number of IFN-γ-secreting CS-specific CD8+ and CD4+ T-cells were determined by ELISPOT assay it was found that co-administration of α-GalCer increased the number of IFN-γ-secreting CS-specific CD8+ cells seven fold. Thus, the overall amplification of the adaptive immune response by iNKT cells made them very attractive adjuvant targets.
Subsequently, α-GalCer has been demonstrated to have adjuvant properties for influenza, HIV, and tumor vaccines in mice (Huang, Y., et al., Enhancement of HIV DNA vaccine immunogenicity by the NKT cell ligand, α-galactosylceramide 2008 Vaccine 26:1807-1816; Ko, S. Y., et al., α-Galactosylceramide can act as a nasal vaccine adjuvant inducing protective immune responses against viral infection and tumor 2005 J. Immunol. 175: 3309-3317; Seino, K., et al., Natural killer T cell-mediated antitumor immune responses and their clinical applications 2006 Cancer Sci. 97: 807-812).
Furthermore, because α-GalCer was discovered while screening for compounds with anti-tumor properties, it has been used in several clinical trials in cancer patients. Delivery by pre-loading autologous PBMCs with α-GalCer in vitro or by direct injection, α-GalCer was shown to be safe and well tolerated. However, although modest enhancement of immune responses was generally seen, its beneficial effects were limited (Giaccone, G., et al., A phase I study of the natural killer T-cell ligand α-galactosylceramide (KRN7000) in patients with solid tumors 2002 Clin. Cancer Res. 8:3702-3709; Ishikawa, A., et al., A phase I study of α-galactosylceramide (KRN7000)-pulsed dendritic cells in patients with advanced and recurrent non-small cell lung cancer 2005 Clin. Cancer Res. 11: 1910-1917; Nieda, M., et al., Therapeutic activation of Vα24+Vbeta11+ NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity 2004 Blood 103: 383-389).
Consequently, Tsuji and colleagues made an effort to find analogues of α-GalCer with increased CD1d-binding and iNKT-stimulatory properties. The particular advantages of identifying a new glycolipid adjuvant similar to α-GalCer and based on a CD1d-binding, iNKT-stimulatory effect are multi-fold. First, the phenotype and functional properties of the CD1d molecules and invTCR of iNKT cells have been conserved between humans and mice, thereby allowing prediction of the activity of related glycolipids in humans through mouse studies. Second, α-GalCer itself has been approved and well characterized in terms of safety and activity in humans. Third, related glycolipids used as vaccine adjuvant could be administered in much smaller quantities using a local parenteral route of administration (e.g. intramuscular) than the larger doses of α-GalCer currently dispensed IV for cancer therapy, thereby further minimizing potential systemic side effects.
Tsuji and colleagues screened a library of synthetic α-GalCer analogues and identified glycolipids with far greater CD1d binding and activation of iNKT cells (Li, X., et al., Design of a potent CD1d-binding NKT cell ligand as a vaccine adjuvant 2010 Proc Natl Acad Sci USA 107: 13010-13015; U.S. Pat. No. 7,923,013). One such glycolipid, was 7DW8-5 (FIG. 1). Structurally, 7DW8-5 possesses a fluorinated benzene ring at the end of C10 length fatty acyl chain. It was selected due to its superior ability to elicit cytokine production from human and mouse iNKT cells and its adjuvant properties in mice when used in combination with a suboptimal dose of a recombinant adenovirus expressing P. yoelii CS protein. The adjuvant was co-administered intramuscularly (IM) with the vaccine. The mice were challenged with pathogenic P. yoelii sporozoites 2 weeks later. 7DW8-5 enhanced the malaria-specific CD8+ T cell response significantly more than α-GalCer and also enhanced the malaria-specific humoral response equally if not slightly stronger than α-GalCer. Finally, 7DW8-5 was able to display a significantly stronger adjuvant effect than α-GalCer in enhancing protective efficacy of the adenovirus recombinant vaccine after a single immunizing dose.
The practical considerations for the preclinical and clinical development of 7DW8-5 as an adjuvant for candidate recombinant subunit malaria vaccines was recently discussed (Padte, N. N., et al., Clinical development of a novel CD1d-binding NKT cell ligand as a vaccine adjuvant 2010 Clin. Immunol. doi: 10.1016/j.clim.2010.11.009).
There is a need for improved malaria vaccines. With regard to malaria vaccines whose immunogen is live attenuated Plasmodium parasites, particularly sporozoite-stage parasites, an adjuvant that could reduce the numbers of doses and the dosages of each dose required for highly effective protection would have enormous value in the fight against malaria. For instance, a vaccine administered in 1 or 2 doses would not only reduce the cost of goods to produce it, but more importantly it would simplify the logistics of delivery for travelers, for rapidly deployed military, or for mass-immunization campaigns.